ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens

Abstract

32Background: Real-Time PCR (RT-PCR) is widely used as the gold standard for 33 clinical detection of SARS-CoV-2. However, due to the low viral load in patient 34 throat and the limitation of RT-PCR, significant numbers of false negative reports are 35 inevitable, which should not be ignored. 36 Methods: We explored the feasibility of droplet digital PCR (ddPCR) to detect 37 SARS-CoV-2 from 57 clinical pharyngeal swab samples and compared with RT-PCR 38 in terms of the sensitivity and accuracy. Among 57 samples, all of which were 39 reported as negative nucleic acid by officially approved clinical RT-PCR detection, 43 40 samples were collected from suspected patients with fever in clinic, and 14 were from 41 supposed convalescents who were about to discharge after treatment. The experiment 42 was double-blind. 43 Results: The lower limit of detection of the optimized ddPCR is at least 500 times 44 lower than that of RT-PCR. The overall accuracy of ddPCR for clinical detection is 45 94.3 %. 33 out of 35 negative pharyngeal swab samples checked by RT-PCR were 46 correctly judged by ddPCR based on the follow-up investigation. In addition, 9 out of 47 14 (64.2 %) supposed convalescents with negative nucleic acid test twice by RT-PCR 48 were positive by ddPCR detection. 49 Conclusions: ddPCR shows superiority for clinical detection of SARS-CoV-2 to 50 reduce the false negatives, which could be a powerful complement to the current 51 standard RT-PCR. Before the ddPCR to be approved for diagnosis, the current clinical 52 practice that the convalescent continues to be quarantined for 2 weeks is reasonable 53 and necessary. 54 55

Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals

“…First strand cDNA was synthesized using PrimeScript RT kit (TakaRa). Optimized ddPCR was used to detect the presence of SARS-CoV-2 viruses following our previous study 10 . Analysis of the ddPCR data was performed with QuantaSoft software (Bio-Rad).…”

“…In this study, we sampled airborne SARS-CoV-2 and its aerosol deposition at 30 sites in two designated hospitals and public areas in Wuhan and then quantified the SARS-CoV-2 copy counts of aerosol samples using a robust droplet digital PCR-based detection method (ddPCR) 10 . The two hospitals are exclusively used for COVID-19 patient treatment during the outbreak but each with unique characteristics serving different purposes.…”

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

“…Lu et al report 96.3% accuracy for testing of clinical samples using digital PCR and were able to detect virus in 4 patient samples that were deemed negative by RT-PCR (Lu et al 2020a). Furthermore, digital droplet methods have been shown to be capable of detecting down to 0.4 viral RNA copies/µL in patient samples (Suo et al 2020). Because digital PCR allows for more careful quantitation of viral RNA copy number over the course of the disease, this highly sensitive test may also be useful to evaluate treatment progress or assess patient release after quarantine.…”

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

“…In this context, the droplet digital PCR (ddPCR) might be more appropriate for quantitation of viral loads as reported previously 7 . The ddPCR allows precise quantitation of nucleic acid copies without the need of any calibration curve and with higher resistance to the ampli cation inhibitors compared to the quantitative real-time PCR 8 , and some recent studies reported the usage of ddPCR for the quantitation of SARS-CoV-2 [9][10][11][12][13] . However, all these ddPCR procedures included the RNA extraction/puri cation step leading to potential errors for the ampli cation due to variable and suboptimal nucleic acid yields 14,15 .…”